Abstract: The environmental element selenium (Se) is essential for human health in small amounts, but toxic at high levels. There is strong evidence that even small changes in Se status have multi-faceted effects on human health which include altered immune function and susceptibility to viral infections, biochemical stresses, cancer, and even diabetes. Many of the beneficial effects of Se can be attributed to selenoproteins; proteins containing Se in the form of the 21st amino acid selenocysteine (Sec). The diversity of substrates and biochemical pathways that selenoproteins act upon likely explains the multiple health effects associated with Se intake. Se is incorporated into selenoproteins co-translationally by redefinition of a UGA codon to encode Sec. In mammals UGA-Sec codons are decoded by two isoforms of Sec-tRNA[Ser]Sec differing only by the presence or absence of a methyl group at the anticodon position Um34. Methylation of this residue in vivo is dependent upon Se status. The unmethylated isoform predominates when Se is deficient, whereas the opposite is true under Se sufficiency. Previous studies suggest that the house-keeping selenoproteins can incorporate Sec using either isoform, whereas the stress-related selenoproteins prefer the methylated form of Sec-tRNA[Ser]Sec. We hypothesize that Se-dependent changes in the ratio of unmethylated to methylated Sec-tRNA[Ser]Sec provides the direct link between Se availability and gene-specific regulation of selenoprotein production. To test this hypothesis and further explore translational control of selenoprotein expression in vivo (Aim 1), we will develop and validate ribosome profiling as a methodology to quantify ribosome position and density on selenoprotein mRNAs in tissues from mice raised on Se deficient, adequate or supplemented diets. In addition, we will examine ribosome distribution in tissues from transgenic mice overexpressing the unmethylated isoform of Sec-tRNA[Ser]Sec. In Aim 2, we will establish an in vitro tRNA-defined rabbit reticulocyte translation system programmed with the methylated or unmethylated isoforms of Sec-tRNA[Ser]Sec to directly test Sec incorporation efficiency during translation of a subset of the house-keeping and stress-related selenoprotein mRNAs and identify the cis-acting elements involved. The results from experiments proposed here promise to establish a new methodology to examine selenoprotein synthesis and to further elucidate the mechanism of Se-dependent regulation of selenoprotein expression, a finding of direct relevance to the health effects of Se.